已知致癌Ras蛋白呈现多种构象状态,据报道,各种各样的晶体结构。GTP绑定状态被分组为两个主要状态:\"inactive\"状态1和\"active\"状态2。最近关于H-Ras的报告表明,状态2表现出两个子状态,与Tyr32的方向直接相关:朝向GTP结合的口袋和向外。在本文中,我们表明N-Ras表现出状态2的另一个亚状态,与Tyr32的第三方向有关,朝向Ala18并平行于GTP结合的口袋。我们还表明,该子状态在N-Ras的G12V突变中高度采样,几乎不存在于其野生型形式中,G12V突变禁止GTPase激活蛋白(GAP)结合亚状态的采样,使这种突变致癌。此外,使用分子动力学模拟,我们探讨了膜对N-Ras构象动力学的重要性及其对Ras蛋白稳定性的强烈影响。此外,膜对Ras的构象(亚)态采样有显着影响。这个,反过来,在Ras的激活/失活循环中至关重要,由于鸟嘌呤核苷酸交换因子蛋白(GEF)/GTP酶激活蛋白(GAP)的结合。
Oncogenic Ras proteins are known to present multiple conformational states, as reported by the great variety of crystallographic structures. The GTP-bound states are grouped into two main states: the \"inactive\" state 1 and the \"active\" state 2. Recent reports on H-Ras have shown that state 2 exhibits two substates, directly related to the orientation of Tyr32: toward the GTP-bound pocket and outwards. In this paper, we show that N-Ras exhibits another substate of state 2, related to a third orientation of Tyr32, toward Ala18 and parallel to the GTP-bound pocket. We also show that this substate is highly sampled in the G12V mutation of N-Ras and barely present in its wild-type form, and that the G12V mutation prohibits the sampling of the GTPase-activating protein (GAP) binding substate, rendering this mutation oncogenic. Furthermore, using molecular dynamics simulations, we explore the importance of the membrane on N-Ras\' conformational state dynamics and its strong influence on Ras protein stability. Moreover, the membrane has a significant influence on the conformational (sub)states sampling of Ras. This, in turn, is of crucial importance in the activation/deactivation cycle of Ras, due to the binding of guanine nucleotide exchange factor proteins (GEFs)/GTPase-activating proteins (GAPs).